After wounding of the skin, one of the objectives is to close the defect as quickly as possible. This is especially important in the case of extensive burns, in order to limit infection risk and fluid loss. Apart from several dressing and grafting techniques developed over the past decades, the use of cultured epidermal sheets has gained increasing attention. This technology allows the covering of the wound with a stratified sheet of living keratinocytes, derived either from the patients' own skin (autologous sheets) or from other donors (allogeneic sheets). Interestingly, although it has been shown that allogeneic keratinocyte sheets have only a limited survival time on the acceptor wound, they have the same clinical effect as autologous sheets when used in combination with split thickness meshed autografts or when applied on partial thickness wounds. This has been explained by hypothesising the existence of wound healing factors produced by these keratinocytes. These factors enhance the wound repair process by stimulating the proliferation and/or migration of the various cell types present in the wound. A new autologous epidermis can thus be formed by the outgrowth from keratinocyles present in the meshed skin autografts or epidermal appendages (e.g. sweat glands, hair follicles).
Although clinically successful in many cases, and sometimes lifesaving, cultured keratinocyte sheets suffer from a number of obvious disadvantages. They are extremely expensive, time-consuming to prepare and difficult to transport, apply and preserve for prolonged periods. Therefore, it would be desirable to develop a wound therapeutic based directly on keratinocyte-derived wound repair factors, without the need of using the living cells themselves.
Several growth factors with potential therapeutic interest in the field of wound healing have been identified or isolated already in keratinocytes. These include, among others, TGF-.alpha., bFGF, amphiregulin and HB-EGF. However, to date, none of these factors has proven to provide the same clinical potential as the living keratinocyte cultures.
One factor of particular interest is amphiregulin (AR). This is a heparin-binding glycoprotein of approximately 20 kDa which was originally purified from phorbol ester-treated MCF-7 human breast carcinoma cells. The factor belongs to the EGF-family of growth factors and stimulates the proliferation of several cell types (including keratinocytes and some fibroblast cell lines), while inhibiting the proliferation of other cells (including many carcinoma cell lines) (Shoyab et al., Proc. Natl. Acad. Sci. USA 85, 6528-6532 (1988); Shoyab et al., Science 243, 1074-1076 (1989)). Sequencing of AR revealed the existence of two forms, containing 78 and 84 amino acids, respectively (Shoyab et al, Science 243, 1074-1076 (1989), Plowman et al., Mol. Cell. Biol. 10, 1969-1981 (1990)). The N-termini of these two subforms were reported to be located at residues 101 and 107 of the preprotein sequence (taking the initiation methionine as residue No.1), whereas the C-terminus was reported to be located at residue 184 of the preprotein sequence. Later studies revealed however that the C-terminal processing site is located probably at least 2 amino acid residues downstream from the originally reported one (Adams et al., Biochim. Biophys. Acta 1266, 83-90 (1994); Thompson et al., J. Biol. Chem. 271, 17927-17931 (1996)). This means that the total length of the published amphiregulin subforms should be at least 80 and 86 amino acids, respectively. To avoid confusion, we will henceforth designate the subforms with their respective N-terminal- and C-terminal endpoints (taking the initiation methionine of the preprotein as residue No. 1). Glycosylation seems not to be important for biological activity of the molecule (Shoyab et al., Proc. Natl. Acad. Sci. USA 85, 6528-6532 (1988)). Later studies revealed that a major keratinocyte autocrine factor is also amphiregulin (Cook et al., Mol. Cell. Biol., 11, 2547-2557 (1991); Cook et al., In Vitro Cell. Dev. Biol. 28A, 218-222 (1992); Piepkorn et al., J. Cell. Physiol. 159, 114-120 (1994)). In cultured keratinocyte conditioned media also, two subforms starting at residues 101 and 107 of the preprotein were detected. A special feature of AR is that its biological activity is completely blocked in the presence of heparin sulphate. It has been reported to be the only growth factor displaying this property (Cook et al., Mol. Cell. Biol., 11, 2547-2557 (1991); Cook et al., In Vitro Cell. Dev. Biol. 28A, 218-222 (1992), Piepkorn et al., J. Cell. Physiol. 159, 114-120 (1994)). The isolation, properties, cloning and potential therapeutical use of AR are disclosed in U.S. Pat. No. 5,115,096 (May 19, 1992), assigned to Oncogen (Seattle).